Altered serotonergic transmission and presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) expression have long been associated with psychiatric disorders including depression, suicide, autism, OCD, impulsive violence. Indeed, the drugs that block SERT such as tricyclic antidepressants and SSRIs are successfully used for the treatment of mental disorders. Studies from PI's group, collaborators and colleagues have identified signaling mechanisms of SERT regulation by kinases/phosphatases. Remarkably, the association of human SERT coding variants Gly56Ala, Ile425Val mutation with OCD, autism and other psychiatric disorders, and the discovery that these mutations alter PKG/p38 MAPK regulation of SERT activity suggest that this form of regulation is important in the normal physiology of SERT and dysregulation of SERT may influence risk for disorders attributed to compromised 5-HT signaling. Currently no transgenic animal models are available to test whether altered SERT phosphorylation is causative for altered behavior found in autism, OCD and other psychiatric disorders. In this R21 proposal, recognizing the risk versus reward mission, we propose to test the hypothesis that transgenic mouse models mimicking constitutive SERT PKG-phosphorylation show gain of 5-HT transport phenotype with loss of PKG-mediated upregulation and phosphorylation rescuing behavioral phenotypes that parallel Ile425Val mutant identified in OCD and Asperger syndrome. In Specific Aim 1, we propose to construct targeting vectors carrying Thr276Asp mutation that mimic PKG-phosphorylation and Ile425Val mutation associated with OCD and Asperger syndrome by inserting the mutations into mouse SERT genomic sequences to generate SERT transgenic SERT mice. Specific Aim 2 will validate SERT regulation and phosphorylation in Thr276Asp and Ile425Val SERT mice, and elucidate important 5-HT related behavioral phenotypes rescued in the knock-in mice. However, future studies that are beyond the scope of this proposal, will utilize Thr276Asp and/or Ile425Val knock-in SERT mice to analyze neurochemical, behavioral and gene expression profiles, and to measure responses to acute and chronic in vivo SSRI administrations and other therapeutic agents. Thus, generation of these unique mouse models provide innovative tools for exploring the kinase mediated SERT regulatory pathways that are set points in disrupting normal SERT function found in disease-linked human SERT variants. In addition, these SERT transgenic mice will aid future studies exploring serotonin-related gene regulatory network that may be linked to mental disorders and in the development of effective pharmacological agents for the treatment of mental disorders. PUBLIC HEALTH RELEVANCE: The association of human SERT coding variants Gly56Ala, Ile425Val mutation with OCD, autism and other psychiatric disorders, and the discovery that these mutants alter PKG/p38 MAPK regulation of SERT activity suggest that this form of regulation is important in the normal physiology of SERT. The proposed research to generate a knock-in mouse model will enable to identify the neuronal network linked to mental disorders and aid in the development of effective pharmacological agents for the treatment of mental disorders and other disease states resulting from aberrant monoamine transmission.